PROJECT SUMMARY Brain tumors have become the leading cause of cancer-related death in children. Ependymoma (EPN) accounts for a substantial number of these deaths, and unlike in medulloblastoma, no effective chemotherapy has been identified. Most pediatric ependymomas, unlike in adults, occur in the posterior fossa. While aggressive surgery and radiation improve the initial results but even in completely resected and radiated posterior fossa ependymoma the 10-year progression survival is very poor with only approximately one third of these children being without relapse. All relapsed children with ependymoma will relapse again and all will die, often after multiple relapses with damaging repeated surgeries and radiation. There is a desperate need to understand the biology of these tumors better to understand the high, and often delayed, relapses. Bulk examination of childhood ependymoma samples has allowed classification within ependymoma which has had identified subpopulations with early relapse risk but has had no impact on therapy or long-term outcomes. Given that relapses are late, seen most frequently in children who have had complete surgeries and are radiated, we hypothesized that relapses occur from a relatively small number of resistant cancer stem cells present at diagnosis. Preliminary data using single cell RNA seq on posterior fossa ependymoma strengthens this hypothesis and identifies a putative cancer stem cell subpopulation amongst 4 distinct subpopulations. This grant rigorously explores whether we have indeed identified a cancer stem population in childhood ependymoma. Our research intends to fully characterizes the distinct ependymoma subpopulations identified in single cell RNA seq. Potentially targeting a stem cell population at diagnosis in addition to surgery and radiation may improved outcomes for a pediatric brain tumor that has substantial mortality.